Epigenetic modifications of regulatory elements on chromatin profoundly impact gene expression and play a role in processes such as cellular differentiation and transformation. These post-translational modifications occur on DNA and histones and are mediated by epigenetic modifying proteins. The Polymerase Associated Factor complex (PAFc) is an epigenetic modifying complex necessary for the deposition of several epigenetic modifications associated with transcriptional activation (including H2Bub, H3K4me and H3K79me). We have recently shown that the PAFc is essential for human leukemias harboring rearrangements of the Mixed Lineage Leukemia (MLL) gene as well as several other subtypes of acute myeloid leukemia (AML). Our preliminary data shows the PAFc is also necessary for fetal hematopoiesis. Further, we found the PAFc is regulated by interaction with the H3K9 methyltransferase SETDB1, which suppresses leukemic transformation. Objective: The role of the PAFc in hematopoiesis and the distinct epigenetic functions required for cellular transformation remains unclear. Our preliminary studies have revealed a novel interaction between the PAFc and an H3K9 methyltransferase, SETDB1 and a role for the PAFc in fetal hematopoiesis. We hypothesize that the PAFc-SETDB1 interaction promotes hematopoietic differentiation and that interference of this interaction aids in transformation by promoting transcriptional activation of a gene program blocking differentiation. Specific Aims: We aim to (1) Characterize the role of the PAFc subunit, Cdc73, in hematopoiesis, (2) Validate the role of the PAFc interaction partner, SETDB1, as a hematopoietic tumor suppressor and (3) Define the mechanism by which SETDB1 modulates PAFc mediated transcriptional activation. Study Design: We have developed a mouse model that allows for the conditional deletion and subsequent characterization of the PAFc subunit, Cdc73, in adult hematopoietic tissues. We will use mutants of CDC73 that alter interaction with SETDB1, as well as overexpression and knock down to evaluate the role of SETDB1 and the PAFc-SETDB1 interaction in differentiation. We will also use a combination of biochemical, molecular and high throughput methods to query the transcriptional consequences of SETDB1 interaction with the PAFc. Health Impact: Epigenetic modifiers are recognized as critical players in cellular differentiation. They also play important roles in hematologic disease and have been validated as viable therapeutic targets. To understand processes like hematopoietic differentiation and transformation, we must define the mechanisms regulating epigenetic modifiers. As the PAFc plays a role in hematopoiesis and several diseases, the proposed research will reveal the role of a previously uncharacterized epigenetic regulator complex in hematopoiesis while also defining how protein interactions, like SETDB1, module the PAFc function during hematopoietic differentiation.